Myosin synthesis in cultures of differentiating chicken embryo skeletal muscle

DEVELOPMENTAL

Myosin

BIOLOGY

29, 113-138 (1972)

Synthesis

in Cultures

of Differentiating

Skeletal Department

Chicken

Embryo

Muscle

BRUCE PATERSON

AND R. C. STROHMAN'

of Zoology,

University

of California,

Accepted

March

Berkeley,

California

94720

20, 1972

Myosin synthesis rates have been measured in cultures of differentiating chick embryo muscle cells before, during and following a period of rapid cell fusion leading to the formation of functional striated muscle. Prior to this fusion “burst” there is a low level of myosin synthesis attributable to a small number of multinucleated myotubes normally present in the culture by 24 hr. This low level of synthesis is not affected by bromodeoxyuridine at concentrations known to block myogenesis. Myosin synthesis begins at a linearly increasing rate with a lag period of approximately 4 hr following the main fusion burst. Cell fusion is reversibly blocked by addition of ethylene-glycol-bis(aminoethy1 ether)N,N’-tetraacetic acid (EGTA) to the culture medium. EGTA has a high affinity for calcium ions and the EGTA-fusion block is reversed by addition of calcium. EGTA does not interfere with myosin synthesis once it has been initiated in control cultures. Myoblasts will divide in the presence of EGTA at least once. By 60 hr in EGTA cultures, the myoblasts have ceased dividing and are arrested in G, or G, of the cell cycle. On the addition of calcium, these cells fuse with an average rate of 10% per hour and complete the main fusion burst within 6 hr. EGTA-fusion-blocked cells do not synthesize myosin until fusion is permitted by calcium addition. The lag period for myosin synthesis in these released cells is approximately 4 hr longer than for control cultures. While EGTA-fusion-blocked cells cannot synthesize myosin, they have differentiated to the point of being competent to fuse. Such cells will fuse on calcium addition in the presence of actinomycin D or cycloheximide. It is tentatively concluded that, in the regulation of chicken skeletal muscle myogenesis, cell fusion potential and the potential for myosin synthesis are developed not simultaneously, but sequentially.

single cells are produced with contractile myosin and actin filaments. For cells The exact timing of contractile protein synthesis in the development of chick derived from 12-day skeletal muscle, myoembryo striated muscle remains unre- sin is not detected until 18-24 hr after solved. The studies of Holtzer and his the time when the majority of myogenic cells undergo cell fusion (Coleman and collaborators (see Okazaki and Holtzer, 1966) have made it clear that presump- Coleman, 1968). For the somite cells, tive myoblasts in culture undergo a ter- the appearance of myosin filaments follows approximately lo-12 hr after the minal mitosis prior to the time of appearterminal S period of the presumptive ance of myosin and actin filaments. Two myoblast (Okazaki and Holtzer, 1966; separate cases need to be distinguished. Holtzer, 1967). In both cases therefore, First, in cells derived from 12-day chick embryo skeletal muscle, the myoblasts one involving cell fusion and one indepennormally fuse prior to the appearance of dent of cell fusion, a considerable lag myosin filaments. In the second case, period exists between the appearance of cells derived from early chick embryo myosin and the time of withdrawal of the somites do not fuse prior to detection of presumptive myoblast from the mitotic myosin filaments, but remain in an ex- population. In both cases, however, the tended G, or G, period. In the latter case, methods used for the detection of myosin lacked the sensitivity required for the ’ To whom correspondence should be addressed. critical analysis of the lag period within INTRODUCTION

113 Copyright All nphta

0 1972 by Academic Press, Inc. of reproductwn ,n anv form reswred

114

DEVELOPMENTAL

BIOLOGY

which the regulatory events for myosin synthesis are presumably unfolding. We have reexamined this lag period for cells derived from 12-day chick embryo skeletal muscle using an assay system for myosin capable of detecting the synthesis of approximately 100 myosin molecules per cell equivalent per hour. The cellular system which normally fuses prior to myosin synthesis was chosen because it is possible to obtain culture conditions in which fusion may be regulated and largely synchronized (Hauschka and Konigsberg, 1966; Coleman and Coleman, 1968; O’Neill and Strohman, 1969; O’Neill and Stockdale, 1972). In addition, as pointed out by Okazaki and Holtzer (1966) and Holtzer (1970a, b), the myoblast prior to cell fusion has had to repress pathways leading to DNA synthesis and presumably then, following fusion, derepress pathways leading to myosin synthesis. The synchrony of development possible in cultures of fusing myoblasts should permit a biochemical analysis of this repression and presumed derepression. This study reports the kinetics of myosin synthesis in normal cultures before, during, and following the main burst of cell fusion which has a duration of approximately 12 hr. In addition, cell fusion is blocked by addition of EGTA to the cellular milieu and the block is released by addition of calcium. The effect of this fusion block on myosin synthesis is studied together with the effect of EGTA on other aspects of myoblast synthetic activity. EGTA appears to have minimal effects on normal synthetic activity except to block myoblast cell fusion confirming the work of Shainberg et al. (1969, 1971) on calcium depletion effects in cultures of rat myoblasts. The increased synchrony for cell fusion on release of the block permits a critical examination of the relationship between cell fusion and the regulation for myosin synthesis.

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29, I%'2

MATERIALS

AND METHODS

Cultures. Primary cultures of 12-day chick breast muscle were prepared as described previously (O’Neill and Strohman, 1969). Labeling conditions. Culture dishes were drained as thoroughly as possible of all residual complete medium leaving a maximum of 0.2 ml of medium on the largest culture dish used. Where relevant, all data reported include corrections in the specific activity of the leucine isotope due to residual complete medium on the culture dish. All short-term labeling, 1 hr or less, was carried out in leucine-free MEM in a volume of 3.0 ml for 100 mm culture dishes and 1.0 ml for 60-mm culture dishes. Long-term labeling and ?a labeling were carried out in complete medium. All media were temperature equilibrated (37°C) before addition to the cultures. 4,5-H3 leucine (30-50 Ci/ mmole), UL 14C-leucine (250 mCi/mmole), 3H-methylthymidine (15 Ci/mmole), and ‘5Ca (3-40 Ci/gm) were purchased from New England Nuclear and were used at isotope concentrations listed in the figure legends. Each batch of leucine used was checked for isotopic purity and content on cellulose thin-layer plates according to Pataki (1971). The isotopic content of all samples was analyzed on a Nuclear Chicago Mark-I counter by channels ratio with external standard. 3H and 14C were counted with 30-35% and 72-75% efficiency, respectively. Inhibitors. Actinomycin D (2 pg/ml), cycloheximide (10 pg/ml) and bromodeoxyuridine BrdUrd (5 yg/ml) were used in complete medium at the times indicated in the figure legends, and were purchased from CalBiochem. EGTA fusion block. Stock solutions, 50 mM, of EGTA were made up in either water or calcium-magnesium-free PBS adjusted to pH 7.4 with 5 N NaOH and

PATERSON AND STROHMAN

Myosin

sterilized by filtration through a 0.20-p pore Millipore filter. Stocks were stored at 4°C and kept no longer than 1 month. The exact concentration of EGTA required to stop fusion varies with each batch of serum and embryo extract and must be determined empirically. For the experiments reported here a range of 1.80-1.95 mM final concentration was used. To add the EGTA to the cultures at the appropriate times, medium from all the dishes to be treated was pooled, mixed with the necessary amount of stock EGTA and added back to the cultures. Cultures were released from EGTA arrest in several ways with identical results; by the addition of fresh complete medium, 48-hr conditioned medium, or adjustment of the calcium concentration in the EGTA medium to 2.5 mA4 with a 100 mM stock solution in phosphate free saline. The last method was used routinely for the experiments reported here as it was the most convenient and promoted the least single cell overgrowth on the cultures. Automdiogmphy. After fixation in acetic acid-ethanol (1 part glacial acetic acid to 3 parts 100% ethanol) or Bouins’ fixative, the cultures were taken through a dehydration series from 70% ethanol to 100% ethanol and air dried. The dishes were dipped in 40°C Kodak NTB-II emulsion that had been diluted 1: 1 with water, airdried in a light-tight box with a blower, and developed in light-tight boxes at 4°C for l-2 wk in the presence of desiccant. The dishes were developed on ice for 10 min with ice cold undiluted Kodak D-19B developer, rinsed with water, and fixed for 10 min with Kodak F-5 fixer. The dishes were stained for 2-5 min with Harris’ hematoxylin and developed with ammonia water (a few drops of concentrated NH,OH in 100 ml of water). Cell counts and fusion index. Cell counts to determine cell densities and rates of fusion were made on cultures

Synthesis

in Muscle

Development

115

fixed and stained as for autoradiography. On occasion some cultures were pretreated with ribonuclease (50 pg/ml in PBS) after fixation for 30-60 min at 37°C prior to staining in order to eliminate cytoplasmic background which interfered with nuclear scoring. For each visual score of fusion a minimum of 1000 nuclei were counted in randomly selected fields. Overstaining was the only ground for the rejection of any particular field for scoring. The rates of cell fusion were also determined on the basis of the decline in DNA synthesis during cell fusion as measured by the decreased incorporation of “Hthymidine per microgram of DNA in the culture, hereafter referred to as the drop in normalized thymidine incorporation. The validity of the technique is discussed by O’Neill and Strohman (1969) and O’Neill and Stockdale (1972) and is applicable at the cell densities used in these experiments. Determination of DNA. DNA was determined using the fluormetric method as outlined by Hinegardner (1971). Calf thymus DNA was used as a standard and prepared according to DeDeken-Grenson and DeDeken (1959). In earlier experiments the method of Keck (1956) was used with similar results but less sensitivity. Polysome isolation and display. Polysomes were pulse labeled with 3H-leucine for 15-60 min and were extracted and displayed on 15-40s (w/v) linear sucrose gradients as described by Hosick and Strohman (1971). Cultures were not fed, however, prior to polysome isolation. Measurement activity. Cells

of leucine pool specific

chilled on a slurry of crushed ice were rinsed one to three times with ice cold PBS and were scraped and homogenized in 2 ml of low salt buffer (LSB 0.02 M NaCl, 0.002 M potassium phosphate buffer, pH 6.8), with 50-100 strokes of a Dounce homogenizer (7 ml size, Kontes Glass Co.) fitted with the A

116

DEVELOPMENTAL

BIOLOGY

pestle. Insoluble debris was spun out in the IEC refrigerated centrifuge, Model PR-2, at top speed for 20 min. The supernatant solution, containing the free leutine, was mixed with a fifth of a volume of 50% trichloroacetic acids (TCA) (w/v) and should be kept at 4°C for a minimum of 4 hr. The precipitated material was centrifuged out of solution and the supernatant solution was extracted four times with two volumes of anhydrous ether to remove all the TCA. The extracted fluid was evaporated to dryness in a vacuum oven at 50°C overnight and resuspended in 0.20 ml of glass-distilled water for the pool assay. Greater than 98% of a known amount of 3Hleucine added to a homogenate could be recovered in this process. The leucine in the extract was measured using the ultrasensitive aminoacylation isotope dilution method described by Rubin and Goldstein (1970). In order to check for the conversion of labeled leucine during a pulse an aliquot of the TCA-free solution containing the acid soluble fraction from the cells (the amino acid free pool) was desalted on BioRad AG 5OW-X8 (200-400 mesh) hydrogen form and eluted with 3.5 N NH,. The eluent was vacuum dried, resuspended in 0.1 M HCl, and spotted on cellulose thin-layer plates as described previously. Counts traveling with the leucine carrier were compared to the counts spotted to determine the percent conversion. In addition, the low salt buffer (LSB) soluble proteins extracted from pulsed cells were hydrolyzed in 6 N HCl under vacuum at 110°C for 24 hr, diluted and run on thin-layer plates to determine if all the counts incorporated into protein were attributable to 3H-leucine. With the labeling periods used here there was no appreciable conversion (less than 2%) of leucine during a pulse and all of the counts in nascent protein were in 3H-leutine. Myosin detection in the cultures. Myo-

VOLUME

29, 1972

sin kinetics were based on a standard 1-hr leucine pulse at 10 pCi/ml in leucinefree MEM in all instances. Pulsed dishes, containing approximately 3 to 6 x lo6 cells, were rinsed twice with ice cold glucose-free PBS while resting on a slurry of water and crushed ice. The dishes were scraped with a plastic spatula into a total of 2 ml of low salt buffer (LSB is 0.02 M KCl, 0.002 M potassium phosphate buffer, pH 6.8), and were homogenized with 50100 strokes of a Dounce homogenizer fitted with the A pestle. The tissue was extracted for a minimum of 1 hr with no difference in yield for longer extraction periods of up to 12 hr. The tissue fragments were spun out of solution in 15 ml conical bottom test tubes at top speed at 4°C in the IEC model PR-2 centrifuge for 20 min (2000 g). The supernatant solution was decanted for the leucine pool assay, and the tissue pellet was washed with 2 ml of the LSB buffer without resuspension. Depending upon the initial cell concentration, the pellet was extracted with 50100 ~1 of high salt buffer (HSB, 0.02 M sodium pyrophosphate, 1 mM magnesium chloride, pH 9.5) for 1 hr as described by Baril et al. (1966). The extract was then spun at 105,000 g in the Spinco Model L ultracentrifuge in lo-ml Oakridge tubes (Nalgene) for 30 min using the 40 rotor. The supernatant solution containing the myosin was mixed with 50 pg (5 ~1) of column purified carrier chicken myosin in HSB buffer and was electrophoresed on SDS acrylamide gels. Myosin was prepared as described previously (Paterson and Strohman, 1970). Gel electrophoresis. Gels were prepared according to Paterson and Strohman (1970) except that Tris . glycine buffer was replaced by a Tris .EDTA-borateSDS buffer, pH 8.3 (TEBS-8.3) as resolution could be improved (Peacock and Dingman, 1968). The final solution from the myosin extraction was either sulfonated or mixed with SDS and P-mer-

PATERSON AND STROHMAN

Myosin Synthesis in Muscle lleuelopment

captoethanol to a final concentration of 1.0% each, and heated in foil-capped test tubes in boiling water for 2 min. Both methods gave identical results. The gels were stained with Coomasie Brilliant Blue (0.060 gm in 400 ml of 50% methanol and 50 ml of glacial acetic acid) at 70°C for l-3 hr and destained with 7.5% acetic acid-5% methanol at 90°C with one change. The destained gels were then equilibrated with a solution of 10% glycerol in water (v/v) at 90°C. The slightly swollen gels were frozen in a Dry-Ice acetone bath and sliced into l-mm slices with a JoyceLob1 gel slicer, completely through the myosin region in the gel. This required a maximum of five slices to clearly establish a true myosin peak. A gel ready for slicing is shown in Fig. 1. The gel slices were placed in counting vials, and 0.70 ml of NCS-water was added (90% NCS-10% water v/v) (Basch, 1968). The vials were heated to 50°C for 2 hr, then mixed with 10 ml of standard toluene scintillant and allowed to equilibrate for 2 hr prior to counting. The Coomassie Blue in the gel slice was used as a correlation check on the myosin peak counts and gave no quench in the prepared sample when compared to a directly sliced, unstained gel. Amido black or naphthol blue-black quenches very strongly if used in the same way. 3H-Leucine was counted as previously described. Protein determinations. Protein was measured using the microbiuret assay described by Zamenhof and Chargaff (1957). Bovine serum albumin was used as a standard and was dissolved in 0.1 N NaOH. Protein was determined from the TCA-precipitable material redissolved in 1 N NaOH. This assay can be used easily over a protein concentration range of lo300 pg of protein per assay. Electron microscopy. To prepare the cells for thin sectioning, the medium was decanted and immediately replaced with 2.5% glutaraldehyde in 0.1 M cacodylate

FIG. 1. A Five l-mm hand (heavy to determine

117

standard myosin gel just prior to slicing. slices are taken through the stained H200,000 MW subunit of myosin) in order a true peak.

buffer, pH 7.2. After fixation for 20 min at room temperature, the monolayers were rinsed in 0.15 M cacodylate prior to post fixation in 1% 0~0, in 0.1 M cacodylate, pH 7.2, for 25 min. Following a brief buffer rinse and rapid dehydration in an ethanol series, the cells were embedded in situ with Shell Epon 812. Polymerization was accomplished after 48 hr at 60°C. The Epon was then removed from the culture dish by cooling on Dry-Ice. Single cells were selected with a Leitz slide marker and a micromanipulator, re-embedded on prehardened Epon blocks, and sectioned with a Porter-Blum MT-2 ultramicrotome. Sections were collected on uncoated grids and poststained with uranyl acetate and lead citrate prior to examination with a Seimens ElmiskopI operating at 80 kV. Electron microscopy was provided by Mr. Paul Moss.

118

DEVELOPMENTAL

BIOLOGY

RESULTS

Recovery

of Myosin

from the Cultures

The recovery of myosin from the cultures was determined in the following way. Four 72-hour, fused cultures on loo-mm dishes were given the standard leucine pulse for myosin synthesis. Two of the plates were treated with 0.1% trypsin in PBS to remove the cells as quickly as possible with minimal damage. The cells were then washed twice with 20% horse serum in PBS, pelleted and extracted directly in 100 ~1 of HSB buffer containing 1.0% Triton X-100, for the normal 1 hr extraction period. The detergent completely dissolved all visible cellular membrane material. The remaining two plates were taken through the normal extraction procedure as outlined in Materials and Methods. Subsequent steps in both cases were identical. As shown in Table 1, the total myosin counts recovered in each extraction procedure were identical within the limits of plating variability. Clearly there is very little myosin lost in the low salt wash homogenization step in the standard extraction procedure, and recovery of nascent myosin appears maximal when TABLE EFFICIENCY

Extraction

1. Standard

condition

EXTRACTION

Preparation

extraction”

2. HSB + Triton X-100 detergent), 1 hr 3. LSB, 1 hr HSB, 1 hr Unextracted in HSB 4. LSB, 1 hr HSB, 1 hr Unextracted in HSB

OF MYOSIN

(1%

Two 80.5-hr

cultures

Two 80.5.hr

cultures

Precipitated

“C-myosinb

Precipitated

“C-actomyosinb

VOLUME

29. 1972

compared to direct extraction. Cells could not be directly extracted in SDS as the solubilization of the chromatin produced a solid gel from which myosin extraction was difficult. The wash and extraction buffers were also checked for their ability to extract precipitated purified myosin and synthetic actomyosin. ‘“C-labeled, chromatographed chick myosin was precipitated alone or with column purified G-actin to form actomyosin. 14C counts were not extractable from either pellet during a 1-hr extraction with the low salt buffer (LSB). In addition, less than 5% of the total 14C counts in the pellets were precipitable after a 1 hr extraction in high salt buffer (HSB) followed by centrifugation at 105,000 g for 30 min (Table 1). Extraction conditions appear optimal with the procedures used here. It was assumed that once myosin was synthesized it was immediately incorporated into thick filaments, in or near nascent sarcomeres, and had extraction properties very similar to those of myosin within fully differentiated muscle fibrils. Several recent reports confirm the rapidity of thick filament formation and association into newly 1 WITH

LSB

AND

HSB

Myosin cpm recovered in HSB ( 3H or “C) a) b) a) b) a) b) c) a) b) c)

3896 4178 4178 4254 18 6493 320 45 8152 223

BUFFERS

% of total myosin recovered 93% 98% 100% 100% Less than 0.5% 94.7% 4.8% Less than 0.5% 97% 2.7%

a See Materials and Methods. * Column purified chick “C-myosin prepared according to Richards et al. (1967) was precipitated in the presence and in the absence of column purified G-actin prepared according to Rees and Young (1967). The pellets were extracted with the various buffers listed. Unextracted in HSB refers to cpm remaining in myosin or actomyosin pellet following extraction of precipitated protein in HSB.

PATERSON

AND

STROHMAN

Myosin

Synthesis

in Muscle

119

Development

developing sarcomeres (Shimada, 1971; Larson et al., 1969; Allen and Pepe, 1965; Shimada et al., 1967) and strongly suggests there is no free pool of nascent myosin which exists prior to thick filament assembly. Sensitivity

of the Myosin Assay

The lower limit of myosin detectability was calculated on the basis of the amino acid composition of the heavy chain of chicken myosin and the average leucine pool specific activity during the standard pulse for myosin as determined from several experiments. The leucine pool equilibrates within 5-10 min (Fig. 2) under the pulsing conditions used. Eagle and Piez (1962) have reported a similar value for HeLa cells grown in suspension where pools require approximately 15 min to equilibrate. Using a leucine content of 175 (Paterson, 1972) leucines per heavy chain of chicken myosin and an average leucine pool specific activity of 450 dpm per picomole of leucine (range 385-505 dpm per picomole of leucine), a standard pulse as defined in Materials and Methods, and a myosin peak obtained from acrylamide gels corresponding to 30 cpm above background, counted at 30% efficiency, for a total of 6 x lo6 cells (an average density from 45-70 hr for these experiments) corresponds to a synthesis rate of approximately 75 myosin molecules per cell equivalent or less than one-half of a thick filament per cell per hour (Pepe, 1967). Myosin Kinetics and Normal Cultures

Cell Fusion

in

Previous work in this laboratory (O’Neill and Strohman, 1969) has shown that myoblast cell fusion in culture is closely paralleled by a drop in thymidine incorporation into DNA and by a rapid fall in the activity of DNA polymerase. Cell fusion, as measured either by the decreased rate in normalized thymidine incorporation or by visual observation, begins at about 40-

ol’, 0

10

I 20 Pulse

30 +tme

40

50

7 60

(mvwtes)

FIG. 2. Rate of leucine pool equilibration for control and EGTA fusion-blocked cultures given the standard leucine pulse for myosin kinetics: 10 pCi/ ml 3H-leucine in leucine-free MEM for 1 hr.

42 hr in these experiments and is 90% complete within 12 hr (Figs. 3 and 9A). During this period, 70-80s of the cell population undergoes fusion. The remainder of the cells are fibroblasts except for approximately 10% which continues to fuse over a period extending to 90 hr of total culture time (O’Neill and Stockdale, 1972). It is clear, therefore, that the great majority of myoblasts fuse in a single burst of approximately 12 hr. Myosin synthesis was measured before, during, and following the major burst of cell fusion (Fig. 3). The results show the following: (1) Prior to cell fusion there is a low level of increasing myosin synthesis which is most probably related to the constantly observed small percentage of myotubes in prefusion primary cultures. These myotubes originate as small multinucleated cells which plate together with single cells when the culture is established and/or they arise by early cell fusion in a small percentage of the total initial inoculum (Fig. 4). (2) After approximately 4-6 hr after the onset of the fusion burst, the rate of myosin synthesis begins to increase linearly. This is a true increase in synthesis since the measurement, at each time point, is the incorporation of leucine-3H into the 200,000 MW myosin subunit during a standardized 1-hr pulse. In addition, as discussed below, it is shown that the

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detach cells from ture dish. Myosin Synthesis Blocked Cultures

Hours

after

plallng

FIG. 3. Myosin synthesis and fusion kinetics in normal cultures. Companion cultures were assayed for myosin content, a decrease in normalized thymidine incorporation, and a visual score of fusion as described in Materials and Methods.

specific activity of the leucine-3H in the cellular pools is not significantly different in the fused and unfused cells. The onset of the linearly increasing rate of myosin synthesis was determined as the intersection of straight lines extrapolated from postfusion and prefusion synthesis points and is indicated by a vertical bar in all the myosin kinetics figures. (3) After the burst of cell fusion and the beginning of the linear increase in rate of myosin synthesis, the fused cells continue to expand their potential to synthesize myosin (Fig. 5), since the myosin content per fused nucleus per dish increases throughout the culture period. In fact, the myosin synthesis rate continues to increase out to 90 hr of total culture time. At this time the experiments are normally terminated because the fused cells are cross-striated and spontaneous contractions begin to

the surface in

of the cul-

EGTA-Fusion

It was of interest to see whether cell fusion and myosin synthesis could be separated as independent events in the progress of myogenesis. Presumably if the molecular programs for cell fusion and myosin synthesis were initiated simultaneously following the terminal S-period of the presumptive myoblast-myoblast transition, an appropriate block operating on the fusion mechanism would still permit myosin synthesis in the absence of cell fusion. Recent work in this laboratory (Strohman and Paterson, 1971) and by Shainberg et al. (1969, 1971), Ozawa and Ebert (1970), and Holtzer (1970b) suggested that such an appropriate fusion block could be generated by lowering the external calcium concentration of the muscle culture medium either by medium dialysis or by direct addition of EGTA since this chelator binds calcium very tightly and almost specifically (association constants at pH 7.1; Ca: log,, K is 6.882; Mg: log,,K is 1.092) (Caldwell, 1970). Shainberg et al. (1971) have shown that low calcium fusion arrest simultaneously stops the fusion associated increase in creatine kinase, myokinase, and phosphorylase with no apparent effect on the overall macromolecular syntheses in rat myoblasts, as measured by the incorporation of labeled precursors into DNA, RNA, and protein. Strohman and Paterson (1971) have also reported that the addition of EGTA to the complete medium of embryonic chick muscle cultures will reversibly block fusion and the subsequent rise in the myosin content of the cultures. Upon addition of calcium to the medium the fusion block is immediately reversed, the cells fuse to control levels in less than 6 hr, but do not begin to synthesize myosin at the same rate as post-

PATERSON

AND STROHMAN

Myosin

Synthesis

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121

Deuelopment --

.

’

FIG. 4. The presence of small myotubes in cultures after plating. BrdUrd (5 pg/ml) was added at 8 hr after cells. (2) At 67 hr after plating. BrdUrd as above.

fusion control cultures until 8-12 hr after release. This sequence of events has now been examined in detail. A. Postmitotic Nature Blocked Cultures

of EGTA-Fusion

EGTA added to cell cultures at any time between 20 and 42 hr will block cell

containing bromodeoxyuridine plating; 95% of the total nuclei

(BrdUrd). (1) At 51 hr were in mononucleated

fusion. In this entire series of experiments cell fusion in control cultures begins at approximately 42 hr. Myoblasts will divide at least once with EGTA present from 20 hr in culture. By approximately 68 hr (40-50 hr in EGTA), the myoblasts have practically ceased dividing and have assumed a distinctive attenuated morphol-

BIOLOGY

DEVELOPMENTAL

II

42

I

46

,

50

54 Hours

5s after

62

,

,

66

70

plating

FIG. 5. Rates of myosin synthesis per fused cell in culture, based on the data presented in Fig. 3. The percent fusion was determined from the normalized thymidine drop. The number of fused cells per dish was derived from the percent fusion, the DNA content per dish, and a DNA content per chick cell of 2.2 x IO- I2 gm DNA per nucleus.

ogy with cell lengths of up to 1 mm (Figs. 7 and 8). Fibroblast morphology is not affected even after 78 hr of exposure to levels of EGTA that block myoblast fusion. Fibroblast mobility, morphology, and capacity to divide were examined in wounded confluent tertiary cultures of chick tibroblasts in the presence and in the absence of EGTA. Cells in EGTA migrated into the wounded area and were found dividing along its edge just as in control cultures. There was no detectable difference in cellular morphology in either case (Fig. 6). No effort was made to quantitate the rate of cell migration or the percentage of dividing cells adjacent to the wounded area. This constant morphological differ-

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1972

ence between myoblasts and tibroblasts in arrested cultures was used as a marker for the ultrastructural examination of fusion arrested myoblasts. Electron micrographs of EGTA-treated cells, presented in Fig. 7, reveal the presence of numerous large polyribosomes, all classes of subcellular filaments, including microtubules, a normal complement of subcellular organelles with standard morphologies and no noticeable differences in the gross membrane morphology or structure when compared to control cultures. No myosin filaments could be found in the EGTA-fusion blocked cells we observed. If such EGTA-fusion blocked cultures are released by calcium addition, the subsequent fusion rate is extremely high and the process appears synchronous (Figs. 8 and 9B). This suggested that the EGTA cells were being arrested in a G, or G, state of the cell cycle since fusion is restricted to this period (Bischoff and Holtzer, 1969). The following experiment was therefore performed. Cells were fusion-blocked at 30 hr with the addition of EGTA to a final concentration of 1.93 mM. The cultures were released at 68 hr by addition of calcium to a final concentration of 2.5 mM, and were fixed for autoradiography at 78 hr. Between addition of EGTA and final fixation, the cells were exposed continually to SH-thymidine for four separate time periods; 30-78 hr, 42-78 hr, 52-78 hr, and 68-78 hr. The fixed and developed cultures were then scored for labeled nuclei in myotubes. The cultures in all cases were approximately 65% fused at the time of fixation. As shown in Fig. 10, for cells exposed to 3H-thymidine from 30 hr, 89% of the myotube nuclei were labeled. Thus, 89% of the myoblasts which ultimately fuse by 78 hr had divided at least once in EGTA after 30 hr in culture. By 42 hr, the number of myoblasts undergoing division had dropped to 32%; by 52 hr the decline was

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Synthesis

in Muscle

Development

123

FIG. 6. Fibroblast morphology in the presence and absence of EGTA, and, fusion of EGTA-fusion blocked myoblasts in the presence of inhibitors upon the addition of calcium. (1) Fibroblasts migrating and dividing in the wounded area of a confluent fibroblast culture exposed to 1.93 mM EGTA for 3 days prior to wounding. Cultures were maintained in EGTA after wounding. Note the characteristic fibroblast morphology and the dividing cells (M). (2) Conditions as in (1) except there is no EGTA present. (3) EGTA fusion-blocked cells exposed to 2 kg/ml of actinomycin D 1 hr prior to the addition of calcium. Cells were fixed 3 hr after release in the presence of inhibitor. (4) Conditions as in (3), but using 10 pg/ml of cyclohexamide. (5) EGTA fusion blocked cultures 3 hr after the addition of calcium in the absence of inhibitors.

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FIG. 7. Ultrastructure of an EGTA fusion-blocked cell in a 70-hr culture. EGTA was added at 24 hr. though EGTA fusion blocked cells are extremely attenuated, subcellular morphology is apparently norn Note the presence of microtubules (WI’), microfilaments (MF), large polysomes (fl, several mitochond (m, Golgi apparatus (G), and vacuoles ( W. No thick filaments (myosin) could be found in any of the EG fusion-blocked single cells we examined. (1) x 8100. (2) x 49,500. Microtubules (MT) are 200 A in diamet (3) x 49,500. Microfilaments are 50-70 A in diameter. Inset: Typical EGTA fusion-blocked cell.

Allal. lria TA ;er.

PATERSON AND STROHMAN

Myosin

to 18%, and by 68 hr only 6% of the myoblasts were to undergo division prior to fusion as measured at 78 hr. It is concluded therefore that the myoblasts in EGTA constitute a postmitotic population at the time of calcium release and that roughly 94% of the total myoblasts population is in G, or G, at this time. The other very interesting outcome of this experiment is that even though the EGTA-myoblasts do not fuse, they evidently remove themselves from the mitotic population at the same approximate time and at the same approximate rate as control cells that are fusing in the absence of EGTA. Control cultures begin the burst of fusion at 42 hr and the EGTA-fusion arrested cultures at 42 hr show that 68% of the myoblasts have ceased dividing. The DNA growth curves for control and for EGTA cultures also reflect a comparable rate of DNA synthesis during the fusion and postfusion period (Fig. 11). In order to determine if fusion arrested cells had made all the preparations for fusion prior to calcium release, or, if release triggered the subsequent synthesis of those macromolecules essential for fusion, EGTA-fusion blocked cells were unblocked and observed for fusion in the presence of actinomycin D (2 pg/ml) or cycloheximide (10 pg/ml). Cells were blocked at 24 hr with EGTA and released at 65 hr with calcium addition. The inhibitors were added at 64 hr in order to ensure a drug effect. The cultures were fixed for observation at 68 hr. At these concentrations of inhibitors, RNA and protein synthesis were reduced below 7% of the control levels for companion cultures in EGTA (Table 2). After 3-4 hr in the presence of these drugs the cells begin to degenerate, yet even under these stringent conditions the cells are capable of fusing (Fig. 6). Thus it would appear that fusionblocked cells in EGTA have already accomplished whatever synthesis is neces-

Synthesis

in Muscle

Development

125

sary for fusion and need only a sufficient calcium concentration in the medium to fuse. A similar conclusion has been drawn for embryonic rat myoblasts-fusionblocked by low calcium levels (Shainberg et al., 1969). B. Myosin Synthesis rested Cultures

in Fusion Ar-

Having established that EGTA-fusion blocked cells are postmitotic and fusion capable by 64 hr in culture, myosin synthesis was examined at various times following fusion-blockage by EGTA, and release by calcium. If the cultures are fusion-blocked with EGTA any time between 20 and 36 hr and released at times between 60 and 70 hr, the outcome is invariable. Cell fusion begins very soon following the addition of calcium to the medium. The end points are clear and multinucleated myotubes are visible within 2 hr after calcium addition (Figs. 8 and 9B). By 6-7 hr after calcium addition the cultures have achieved 6070% fusion from a background level of approximately 6-10% fusion. This requires an average minimum fusion rate of 10% per hour. The latter figure is the maximum rate of fusion observed by O’Neill and Stockdale (1972) in their normal cultures, which had an average fusion rate of 5% during the main fusion burst. A fusion rate of 10% per hour is not unreasonable for cultures in which the fusion-arrested cells are held in a postmitotic state and essentially synchronized for fusion. The second invariable result of releasing EGTA-fusion-blocked cells with calcium addition is the lag period observed for myosin synthesis (Fig. 12). In contrast to the control cultures in which the lag between initiation of fusion and initiation of linear increase in myosin synthesis was 4-6 hr, when the EGTA-blocked cells are released, the lag for myosin synthesis is increased to 8-10 hr. We do not have any

126

DEVELOPMENTAL

BIOLOGY

VOLUME

29, 1972

PATERSONAND STROHMAN

Myosin

Synthesis

in Muscle

Development

127

FIG. 8. Growth and differentiation of EGTA-treated cultures. (1) 46 Hr after plating. EGTA was added at 24 hr. The first obvious effect of EGTA is the rounding of the majority of the cells in culture. Fibroblasts are not affected. (2) 56 Hr after plating. Many of the rounded cells have assumed a bipolar shape and are beginning to elongate. Note the flattened fibroblasts. (3) 65 Hr after plating. This is the appearance of the cultures at the usual time of calcium addition. Normal cultures are well fused at this time. (4) 67 Hr after plating; 2 hr after the addition of calcium. Fusion appears to begin at the tips of these elongated single cells. (5) 70 Hr after plating; 5 hr after the addition of calcium. Multinucleated myotubes are quite apparent throughout the culture (6) 72 Hr after plating; 7 hr after the addition of calcium. The fusion process is now complete and approximately 65-70’S of the single cells on the dish prior to the addition of calcium have been incorporated into syncytia. (7) 98 Hr after plating. These cells were maintained in EGTA fusion arrest and were fixed at the same time as the culture illustrated in part 8. Note the extreme attenuation. These cells are often over 1 mm in length. (8) 93 Hr after plating; 28 hr after the addition of calcium. Large myotubes are now found throughout the cultures and myosin synthesis has commenced. These cultures are indistinguishable from control cultures of the same age.

128

DEVELOPMENTAL

BIOLOGY

VOLUME

29, 1972

C :

70-

* .? 0

60-

../i---.

E” 50.5 a,

40-

u 2

30-

z ,o

20-

40-

. .

3020-

. ..

z

IO-

,\”

0,

. :,,m .:. IO

20

..

* * 30

o40

50

60

70

80

50 Hours

60

after

70

80

90

IO

20

30

, 40

, 50

I 60

I 70

plating

FIG. 9. Fusion kinetics for various culture conditions. Cultures were fixed and scored as described in Materials and Methods. (A) Normal culture conditions. Data from 15 different cell platings. (B) Calcium release of EGTA fusion-blocked cultures. All points normalized to 65 hr as the release time (arrow). Based on seven different cell platings. (C) BrdUrd-treated cultures. Filled circles: 8 hr after plating 5 ag/ml BrdUrd. Open circles: 8 hr after plating 1 pg/ml of BrdUrd with an additional 1 pglml at 24 hr after plating. Date from seven different cell platings.

information to explain the increased lag time for myosin synthesis. It may be that the EGTA cells are in a G, period prior to calcium addition and the additional time for myosin synthesis reflects the time needed to move from G, to G, of the cell cycle. Unreleased cells do not fuse and do not synthesize myosin substantially above prefusion rates (Figs. 8 and 12). The low level of myosin synthesis in the EGTA-fusion-blocked cultures is the same as that for prefusion control cultures at 40 hr. This low rate of synthesis gradually increases in the EGTA cultures so that by the time of release with calcium (60-70 hr) the level of myosin synthesis is higher than the control cultures at prefusion (about 42 hr). We have been assuming that this low background synthesis of myosin was due to the small number of precocious, multinucleated cells in the culture (Fig. 4). Since this level of synthesis continued even in the presence of EGTA, which blocks the main fusion burst, the assumption is strengthened.

One might assume, however, that the single, fusion-blocked cells in EGTA were entering into myosin synthesis at the lower rate as they became arrested in G, or G, of the cell cycle. We therefore compared the levels of myosin synthesis of the EGTA-fusion-blocked cultures with that taking place in control cultures to which BrdUrd (5 pg/ml) had been added at 22 hr. According to Bischoff and Holtzer (1970), Stockdale et al. (1964), and Coleman et al. (1969) this level of BrdUrd blocks fusion and further differentiation. As indicated in Table 3 the BrdUrdtreated cultures continue to show a low level of myosin synthesis which is comparable to the level of synthesis in the EGTA-blocked cultures just prior to the standard release time of 68 hr. This finding also supports the notion that the low level of myosin synthesis observed in prefusion cultures is due to small numbers of precocious myotubes present in the culture by 24 hr. These multinucleated cells are expected to continue myosin synthesis in the presence of BrdUrd since their nuclei are no longer making DNA. When

PATERSON AND S.TROHMAN

My&n

Synthesis

in Muscle

Development

6-

GTA + 14 28

32

36

40

44 Hours

L

48

52

after

56

60

64

68

72

76

plating

FIG. 11. Increase in the DNA content per culture in the presence [Cl], and absence [0] of EGTA. Cells were initially seeded at 0.5 x 10’ per 60 mm culture dish in 5 ml of medium. The dashed lines are to emphasize the decreased rate of DNA accumulation per dish during the fusion and postfusion period from 42 to 72 hr. Nate that EGTA fusion-blocked cells do not continue to divide much above prefusion cell numbers in normal cultures.

E

0

30

40

50 Hours

60 after

70

00

plating

FIG. 10. Postmitotic nature of EGTA-fusionblocked myogenic cells. The number in parentheses is the percentage of ‘H-thymidine labeled nuclei in myotubes when label was added at the time point and kept continuously in the medium until fixation. ‘H-methylthymidine (5 &i/ml in complete EGTA medium) was added for the following time periods: 30-78 hr, 42-78 hr, 52-78 hr, and 68-78 hr.

BrdUrd is added together with EGTA, the low level of myosin synthesis is still observed. In addition, we are able to observe, in cultures treated with BrdUrd at 24 hr, that a small percentage of the cells are small, multinucleated myotubes cf. Fig. 4 (see also Bischoff and Holtzer, 1970). It is difficult to avoid the conclu-

TABLE 2 INHIBITION OF MACROMOLECULARSYNTHESISIN EGTA FUSION-BLOCKEDCULTURES BY ACTINOMYCIND AND CYCLOHEXIMIDE~ Actinomycin

Cpm incorporated into RNA or protein

D

Wml)

Uridine

Inhibition (a)

Leucine

1

-

104

-

94

2

-

145

-

92

-

1

-

158

92

-

5

-

93

-

10

-

128 99

95

Control 1871 0.0 Control 1937 0.0 o Cells were blocked with EGTA (1.93 mA4) at 24 hr, treated with inhibitors at 64 hr and released from EGTA-fusion arrest with CaCl, in the presence of either 3H-leucine or uridine (10 &i/ml in complete medium). Cells were pulsed for 1 hr.

130

DEVELOPMENTAL

BIOLOGY

sion that the low levels of myosin synthesis found in these cultures prior to the main fusion burst are accounted for by a small percentage of preformed myo-

FIG. blocked released tained periment). temporary here is plained

after

plating

12. Myosin synthesis in EGTA fusionand released cultures. (O--O, cultures at 60 hr with CaCl, : O---O, cultures mainin fusion arrest for the duration of the exFusion kinetics are given in Fig. 9-B. The rise in the rate of myosin synthesis shown typical of several experiments and is unexat this time.

EFFECTS

OF BrdUrd

TABLE 3 (1.93 m&f),

(5 pg/ml)

AND EGTA PREFUSION LEVELS

Cpm

Hours after plating EGTA 68 92 a Cells

(a) 10,145” were

released

from

OF MYOSIN

in myosin

BrdUrd

(a) 3205 (b) 3101 fusion

block

with

29,

1972

tubes and that the linear increase in myosin synthesis observed results from new synthesis potential gained by cells after they have fused. Finally, experiments were done to answer the criticism that EGTA, while blocking cell fusion, was independently inhibiting myosin synthesis. First, as shown in Table 4, if EGTA is added to postfusion cultures, myosin synthesis continues at the normal rate for at least 4 hr and the synthesis of nonmyosin proteins is not diminished at all. There is a later effect of EGTA. Contractile myotubes in the presence of EGTA begin to detach from the bottom surface of the culture dish so the apparent decrease in myosin synthesis after 6 hr is an EGTA artifact. EGTA does not, therefore, selectively inhibit myosin synthesis once it has been initiated. Second, as shown in Fig. 13, EGTA preincubation will not delay normal fusion and the onset of normal myosin synthesis when prefusion cultures are preincubated with EGTA for 20 hr. In these experiments, the cultures were incubated with EGTA from 22 hr to 42 hr. At 42 hr, the time of initiation of normal cell fusion, excess calcium was added to reverse the effects of EGTA. Myosin synthesis was monitored every 2 hr from 42 to 72 hr. As the results show, myosin synthesis rates for control and EGTA-preincubated cultures were identical within the experimental error. Third, normal cultures were exposed to blocking concentrations of EGTA just prior to the onset of cell fusion at 42 hr. These cultures

D _/----0’Y /Hours

VOLUME

(per

ADDED 23 SYNTHESIS

culture

HR AFTER PLATING,

ON

dish)

EGTA-BrdUrd

Controls

(a) 3057 (b) 3406

(a) 1456 (b) 1654

(a) 13,961 -

(a) 3114

(a) 1812”

(b) 16,045

CaCl,

at 68 hr.

PATERSON

LEUCINE

UPTAKE

AND STROHMAN

Control

(1) 70.5 72.5 74.5 80.5

155,378 164,413 170,708 167,410

Synthesis

in Muscle

TABLE 4 INTO MYOSIN AND NONMYOSIN ABSENCE OF EGTA”

AND INCORPORATION

Hours after plating

Myosin

PROTEINS

cultures’

IN THE PRESENCE AND

EGTA postfusion

(2)

(3)

370 390 383 297

1,131 1,668 2,430 3,900

131

Development

(1) 166,554 133,707 193,561 153,321

treated cultures’.

’

(2)

(3)

394 300 332 275

1,108 1,809 75W 325

D Columns: (1) Total acid-soluble leucine counts per culture; (2) total acid precipitable counts per pg protein per culture; (3) myosin counts per culture. * Cultures 68.5 br after plating were treated with EGTA (1.93 mM). Fusion levels were at the time of addition. ‘The data past 74 hr in these experiments are unreliable and are included to show EGTA on these older, differentiated cultures. By 6-7 hr after EGTA addition to cultures entiated muscle fibers, the fibers become vacuolated and soon thereafter begin to detach The effect is not observed when EGTA is added to cultures prior to myotube formation.

nonmyosin

leucine

approximately

70%

the latent effect of containing differfrom the substrate.

were released by calcium addition at 66 hr, and myosin synthesis was monitored every 3 hr from 66 to 96 hr. As shown in Fig. 14, the EGTA-treated cells, blocked at the time of incipient fusion, do not make myosin until after the release by calcium. When myosin synthesis starts up at the linearly increasing rate, it does not do so until after the full 8-10 hr lag period normally found for EGTA-fusion blocked cultures. This experiment also indicates that myosin synthesis is not initiated just prior to fusion or at the time of incipient fusion. If myosin synthesis were initiated at this time, we would expect to find increased rates of labeling of myosin in the single cells even in the absence of cell fusion. From the above results it is clear that, while EGTA blocks cell fusion, it has no effect on myosin synthesis once it has been initiated and will not prevent normal patterns of myosin synthesis even after a preincubation of 20 hr. 42

46

50

54 Hours

58 after

62

66

70

74

plating

FIG. 13. Myosin synthesis in EGTA blocked cultures released from fusion-arrest time of incipient fusion in normal cultures. was added at 22 hr after plating (O---O, treated cultures; O--O, control cultures). kinetics as in Fig. 9A.

fusionat the EGTA EGTAFusion

C. EGTA Effects Other than Fusion

on

Cell

Function

EGTA appears to have negligible effects on myoblast function other than arresting cell fusion. When prefusion control cultures are compared with EGTA-fusion-

132

DEVELOPMENTAL

BIOLOGY

5k

FIG. blocked onset of calcium

14. Myosin synthesis in EGTA fusioncultures treated with EGTA at 42 hr (the fusion in control cultures) and released with at 66 hr. Fusion kinetics as in Fig. 9B.

blocked cultures, they are found to be comparable in the following respects: (1) the intracellular calcium content, (2) leutine pool specific activity, (3) total protein synthesis, (4) polysome profiles. 1. intracellular calcium content. Preand postfusion control and EGTA treated cultures, pulsed with YZa for the previous hour, were washed out three times with ice-cold Ca-Mg-free PBS containing 10 mA4 EGTA to remove externally bound calcium. Cells were scraped from the dishes in exactly 2 ml of PBS, homogenized, and brought to 5% TCA. After centrifugation of the TCA insoluble material, an aliquot of the supernatant was mixed several times with absolute ether to remove the TCA and was assayed for %a. The DNA content of the cultures was measured on the TCA pellet. As

VOLUME

29, 1972

shown in Table 5, the prefusion and EGTA-treated cultures at 42 hr display insignificant differences in 45Ca content. As fusion proceeds in the control cultures, the ‘%a content also increases reflecting, in all probability, the development of calcium sequestering vesicles of the reticulum. The muscle sarcoplasmic EGTA-treated cells, when released from the fusion block also begin to increase their intracellular calcium content over the prefusion control levels. The main point, however, is that the EGTA-treated cells are not calcium-depleted compared with control cells pre- or postfusion. Evidently, the levels of EGTA used are sufficient to lower the external calcium concentration below a critical level required for cell fusion but do not significantly affect internal calcium concentrations. It should be reemphasized here that the concentrations of EGTA used are critical and must be predetermined for each batch of medium. In these experiments 1.93 mM EGTA was used throughout. If the EGTA concentration is reduced to 1.85 mM, cell fusion is only slightly retarded and reaches control levels within a short time. On the other hand, if the EGTA concentration is raised to 2.0-2.2 mM, the cells detach from the bottom surface of the culture dishes. 2. Leucine pools. The intracellular TABLE 5 SOLUBLE CALCIUM PER CELL EQUIVALENT IN CONTROL AND EGTA-TREATED CULTURES Hours after plating

42 53,. 5 65 72 78

‘%a

cpm

per microgram of DNA

Control cultures

EGTA-treated cultures

294 396 640 1070 1185

260 505 588 1240” 1155”

o Cells were released at 65 hr with CaCl,. Cells were pulsed with *%a at a final concentration of 5 &!i/ml administered to the culture medium. Pulses were for 1 hr prior to removal of cells for assay.

PATERSON

AND

Myosin

STROHMAN

leucine pool specific activity was determined for control and EGTA-treated cultures also being measured for myosin synthesis. Pool specific activity was determined, as indicated in Materials and Methods, by giving leucine-3H pulses to cultures under the standard pulse condition for detecting myosin synthesis. As indicated in Table 6, there are no significant differences between control and EGTA-treated cultures. In addition, it is observed that the specific activity of the leucine pool does not change during cell fusion even though total leucine content per cell equivalent may increase (Table 7). 3. Total protein synthesis. As pointed out earlier (Table 4), EGTA has no effect on myosin synthesis once it has been initiated in normal, fused cultures. As shown in the table, if EGTA is added to fused cultures at 68.5 hours, the total acid soluble leucine, the total amount of leutine incorporated into protein, and the myosin synthesized during a standard pulse remains the same as in sister cultures receiving no EGTA, and continues for 6 hr after EGTA addition. TABLE LEUCINE AND

POOL

SPECIFIC

POSTFUSION CULTURES

Hours after plating

in Muscle

133

Development

4. Polysome profiles and patterns of protein synthesis. Comparisons of polysome profiles from control and EGTAtreated cultures show no significant differences for times prior to the major fusion burst (Fig. 15). While no differences were anticipated here, there was the posTABLE LEUCINE CONTROL

7

UPTAKE PER CELL AND EGTA-TREATED

EQUWALENT IN CULTURES

Hours after plating

Control cultures (pmoles leucine/ pg DNA)

EGTA cultures (pmoles leucine/ rg DNA)

45

48.0

44.0

49

47.0

52.8

55

75.0

62.8

59 69

84.0 148.0

92.0" 80.0"

D Released

from

fusion

block

at 55 hr with

CaCl,.

7

i3

A 6

4

I

6 ACTIVITY

IN PREFUSION

CONTROL AND EGTA-TREATED AFIXR A STANDARD PULSE Control

cultures

Hours after plating

EGTA

cultures”

(dpm Ppicomole leucine) 40 42

Synthesis

436

(dpm per picomole leucine) 67 70

390 390

73 76

480 415

44

520 485

46

560

48 50

580 490

79

410

79b

460

52.5

485

81.5

54.5 67 70

505 425

91

435 405

94 946

465 447

385

a EGTA was added at 24 hr (1.93 were unblocked at 67 hr with CaCl,. tracted from the LSB fraction in the tion procedure. b These cultures were maintained arrest.

mm, and cells Pools were exmyosin extracin EGTA

fusion

Fraction

number

(.-.I

15. Polysomes isolated from control and EGTA-treated cultures after a 20 min pulse with 3H-leucine (10 &i/ml in leucine-free MEM) as described in Materials and Methods. (A) Control cultures 46 hr after plating. (B) Cultures 76 hr after plating treated with EGTA from 66 hr. (C) EGTA fusion-blocked cultures 70 hr after plating, EGTA added at 24 hr. (D) Control cultures 76 hr after plating. Number in parentheses refers to the counts contained in the pellet. Direction of sedimentation is from left to right. FIG.

134

DEVELOPMENTAL

BIOLOGY

sibility that EGTA had some negative effect on larger polysomes and in this way on inhibition of myosin synthesis. No negative effects were found. All classes of polysomes from EGTA-treated cells appeared normal, and the specific activity of nascent polypeptides attached to polysomes from control and EGTA-blocked cultures are comparable within experimental error (Fig. 16). Marked changes in the patterns of synthesis were noted in control and EGTA-treated cultures following cell fusion and the beginning of the linearally increasing rate of myosin synthesis. The specific activity of the large classes of polysomes increased and there was a large increase in labeled protein sedimenting to the bottom of the sucrose gradient (Fig. 15D). We assume that this rapidly sedimenting material represents large polysomes synthesizing myosin. The radioactive protein sedimenting with this material can be completely chased in 15

04 0

IO

20 Polysome

i0 number

40

50

Frc. 16. Specific activity of polysomes isolated from prefusion control cultures 46 hr after plating (O--O) and from EGTA fusion blocked cultures 70 hr after plating (Cl--Cl). Pulse conditions as in Fig. 15. The position of each polysome class beyond 8-mers was determined from a linear extrapolation of a plot of log,, (polysome number) versus distance from the top of the sucrose gradient. Such a plot places 50-70-mers at the bottom of the sucrose gradient in the pellet.

VOLUME

29, 1972

min by following the 3H leucine pulse with fresh medium containing unlabeled leutine. We are currently conducting an examination of the pelleted material. It should be noted here, however, that in our search for myosin-synthesizing polysomes as reported by Heywood et al. (1967) we do not observe the characteristic “hump” of large polysomes near the bottom of the sucrose gradient. This is a minor point, but

calculations

show

that

for this

gra-

dient system, a polysome of 50 or more ribosomes will be pelleted for the duration of the run used here. DISCUSSION

The use of a sensitive assay system for myosin has permitted a systematic analysis of the synthesis of this protein during muscle development in culture. The sensitivity and reliability of the method itself is on solid ground and allows the detection of a myosin synthesis rate of less than one thick filament per cell equivalent per hour, in culture dishes containing 3-6 x lo6 cells. The labeling of the 200,000 MW subunit of myosin with 3Hleucine during a standardized pulse is shown to reflect a true increase in myosin synthesis which is not attributable to differential recovery of myosin (Table l), changes in the rate of leucine pool equilibration (Fig. 2), increase in leucine pool specific activity (Table 6), or problems of leucine conversion (see Materials and Methods). The level of sensitivity for myosin synthesis also takes into consideration the possibility of intracellular leucine pool compartments. Righetti et al. (1971) have presented evidence for leucine pool compartmentalization in HeLa cells. The finding is that newly induced ferritin had a leucine specific activity 30% lower than the free leucine pool specific activity. These authors concluded that amino acids from degraded proteins are capable of circumventing both medium and intracellular free amino acid pools. Hider et

PATERSON

AND STROHMAN

Myosin

al. (1969) have shown that in muscle tissue the amino acids in proteins are incorporated directly from the extracellular pool without dilution through the intracellular free pool. While we have not investigated reutilization and compartmentalization of amino acids in the cultured muscle cells, if we use the figure of 30% as a maximum for the relative decrease in the specific activity of leucine in nascent protein compared to that of the leucine pool, then we calculate a minimum sensitivity for detection of newly synthesized myosin. It is on this basis that we arrive at the figure of less than one thick filament, or about 75 myosin molecules, per cell equivalent (per nucleus) per hour. With this sensitivity in mind, it is clear that cultures of myogenic cells are synthesizing myosin at a low rate prior to the time of the main fusion burst at 42 hr. This is true of control cultures as well as cultures treated with EGTA. The presence of EGTA in the culture medium does not therefore inhibit this low level of myosin synthesis. Small amounts of myosin in prefusion cultures have previously been detected by Coleman and Coleman (1968). They have raised the question of the source of this myosin. Is myosin synthesis taking place at a low level in all myogenic cells of the culture or merely in a few early-forming myotubes which are routinely observed? Since this low level of synthesis continues in the presence of 5 @g/ml of BrdUrd which blocks myogenesis we have concluded that the myosin synthesis detected prior to the main fusion burst is taking place only in cells which have already fused (cf. Fig. 4). Sanger and Holtzer (1970) have reported briefly that myoblasts from lo-day-old chick breast muscle will not fuse in the presence of 1 pg of BrdUrd per milliliter but will form striated mononucleated cells. Sanger et al. (1971) report that myoblasts from the same muscle type will develop cross striations if cytokinesis is

Synthesis

in Muscle

Development

135

blocked with cytochalasin B. These results suggest that fusion in this muscle is not required for synthesis of myosin. The cytochalasin result could lead to the interpretation that any mechanism which leads to multinuclear myogenic cells will allow the synthesis of structural proteins. It does not rule out the requirement for fusion in the normal developmental pathway. The effects of low levels of BrdUrd are more difficult to reconcile with a requirement for cell fusion prior to transcription or translation for myosin. It is not clear, however, what percentage of single cells in the BrdUrd experiments are involved in filament formation. Coleman et al. (1969) using 2.5 pg/ml BrdUrd were not able to observe any. Furthermore, the possibility exists that mononucleated segments of myotubes may be produced by the tissue dissociation procedure used in all of these studies (Fischman, 1970), in addition to small, binucleated myotubes, which persist in the culture. Our preliminary experiments on effects of low levels (1 pg/ml) of BrdUrd indicate that fusion is indeed blocked but we are not able to detect any increase in myosin synthesis beyond the background levels mentioned. A significant level of early fusion is not unusual in embryonic chick muscle cultures and has been reported as high as lo-20% in 24-hr cultures by Okazaki and Holtzer (1966), 5%, by O’Neill and Stockdale (1972), and 58% in the cultures used here. As shown by Dawkins and Lamont (1971), this may be due to small myotubes that are capable of plating out and surviving in culture and can facilitate and supplement low levels of early fusion. Konigsberg (1961) has reported that 14.7% of the cells in his initial cell suspension were binucleated and a very small number, 2%, contained 3-4 nuclei. All taken into account, the existence of a predifferentiated population of cells by 24 hr in culture seems quite clear and this population is

136

DEVELOPMENTAL BIOLOGY

sufficient to account for the prefusion levels of myosin synthesis. If this is so, then the increased rate of myosin synthesis following cell fusion represents a qualitative and not merely a quantitative difference in pre- and postfusion cells in their regulation for myosin synthesis. The lag period for the increased rate of myosin synthesis following cell fusion is between 4 and 6 hr in control cultures. If a time for G, + M of 2 hr is assigned (Marchok and Herrmann, 1967), and the assumption is made that myoblasts are fusion competent immediately after the terminal mitosis (Strohman, 1970; O’Neill and Stockdale, 1972), the lag period between the terminal S period and the rise in rate of myosiri synthesis becomes 6-8 hr. The EGTA studies also indicate that the myogenic cells do not begin a program of translation for myocin immediately after the terminal S-period, that is, during the terminal G, or M period. If this program for myosin synthesis was initiated immediately after terminal S, then one would expect to find a rise in myosin labeling in cultures fusion-blocked with EGTA at 42 hr. At 42 hr, more than 50% of the cells have completed S and are either in terminal G, or M, or have entered the G, or G, of the fusion arrest (Fig. 10). As indicated in Figs. 12 and 14, there is not a rise in myosin synthesis until after the fusion block has been removed by calcium addition. It is concluded that the program for myosin synthesis is not initiated simultaneously with or prior to cell fusion in these embryonic chick skeletal muscle cells. The EGTA-fusion-blocked cells, however, have made some progress toward terminal differentiation following the terminal S period. While these cells show no ability to initiate myosin synthesis, they have evidently acquired the ability to fuse. This conclusion rests on the clear demonstration of cell fusion on the addition of calcium to previously blocked cells in the

VOLUME 29, 1972

presence of either actinomycin D or cycloheximide. Taken at face value, the above experiments suggest that fusion capability is programmed by these cells prior to the initiation of the program for myosin synthesis. The data presented here argues strongly that EGTA has little effect on myogenic cells other than blocking cell fusion. Leutine pool specific activity, intracellular calcium concentration, total protein synthesis and patterns of polyribosomes remain normal in EGTA-fusion-blocked cells. These cells continue to grow as single cells and can reach on overall length of 1 mm. An increase in cell size in the presence of EGTA has been observed by Balk (1970) for normal but not for Rous sarcoma virus-transformed fibroblasts. Under their conditions of long-term incubation with EGTA involving many days the EGTA-treated cells also retained viability and assumed normal proliferative patterns on the addition of calcium ions to the medium. The exact mechanism of the EGTA-fusion arrest is not known, although chelation of calcium ion is clearly involved. Recently, Konigsberg (1971) has shown that a diffusible factor is involved in regulating cell fusion in clones of g-day-old chick embryo muscle. The factor has an apparent molecular weight greater than 300,000. It is entirely possible that such a factor would have a minimal calcium requirement for its activity, and this possibility is testable. The use of EGTA may prove to be a valuable tool. It clearly produces a population of myoblasts synchronized for cell fusion. After addition of calcium to such populations, fusion begins abruptly with a rate exceeding 10% per hour. Temporal separation between cell fusion and myosin synthesis is greatly amplified under these conditions. Myosin synthesis rates do not increase until after a lag period of 8-10 hours. Finally, the data presented here do not

PATERSON

AND STROHMAN

Myosin

show any net increase in polyribosomes following cell fusion. This conclusion was also drawn earlier (Hosick and Strohman, 1971). Since the leucine pool specific activity also remains constant before and after cell fusion, the increased rates of myosin synthesis observed after fusion must be due to increased synthesis or availability of messenger RNA for myosin. The time period for the activation of the events which regulate this mRNA, either at the transcriptional or translational level, is approximately 4 hr in normal cultures. This period can be significantly increased by the use of EGTA to synchronize cell fusion, since the lag time for the synthesis of myosin following release from the fusion block is practically doubled. This increased synchrony of events may prove useful in unraveling mechanisms regulating protein synthesis in this population of differentiating muscle cells. The skilled technical assistance of Alecia Palleroni is acknowledged. Special thanks are extended to Dr. Richard Fluck for the use of unpublished data on fusion kinetics. Supported by U. S. Public Health Service Grant GM 13882, and by U. S. Public Health Service Predoctoral Fellowship (to B. P.) No. l-FOl-GM-42, 709-01.

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